Mitochondria have evolved diverse forms across eukaryotic diversity in adaptation to anoxia. Mitosomes are the simplest and the least well-studied type of anaerobic mitochondria. Transport of proteins via TIM complexes, composed of three proteins of the Tim17 protein family (Tim17/22/23), is one of the key unifying aspects of mitochondria and mitochondria-derived organelles. However, multiple experimental and bioinformatic attempts have so far failed to identify the nature of TIM in mitosomes of the anaerobic metamonad protist, Giardia intestinalis, one of the few experimental models for mitosome biology. Here, we present the identification of a single G. intestinalis Tim17 protein (GiTim17), made possible only by the implementation of a metamonad-specific hidden Markov model. While very divergent in primary sequence and in predicted membrane topology, experimental data suggest that GiTim17 is an inner membrane mitosomal protein, forming a disulphide-linked dimer. We suggest that the peculiar GiTim17 sequence reflects adaptation to the unusual, detergent resistant, inner mitosomal membrane. Specific pull-down experiments indicate interaction of GiTim17 with mitosomal Tim44, the tethering component of the import motor complex. Analysis of TIM complexes across eukaryote diversity suggests that a "single Tim" translocase is a convergent adaptation of mitosomes in anaerobic protists, with Tim22 and Tim17 (but not Tim23), providing the protein backbone.

• MeSH
• anaerobióza MeSH
• Giardia lamblia enzymologie   MeSH
• mitochondrie enzymologie   MeSH
• molekulární evoluce * MeSH
• sekvence aminokyselin MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transportní proteiny mitochondriální membrány MeSH

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway's enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.

• Klíčová slova
• Chromera velia, heterologous expression, predictions, tetrapyrrole biosynthesis,
• MeSH
• Alveolata fyziologie   MeSH
• Apicomplexa metabolismus   MeSH
• biologický transport MeSH
• hem metabolismus   MeSH
• metabolické sítě a dráhy * MeSH
• mitochondrie genetika   metabolismus   ultrastruktura   MeSH
• molekulární evoluce MeSH
• protozoální proteiny chemie   genetika   metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• rozsivky metabolismus   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hem MeSH
• protozoální proteiny MeSH

BACKGROUND: Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. RESULTS: Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) 'classical' Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors ('classical' Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of 'classical' Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. CONCLUSIONS: Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that 'classical' Pufs then underwent lineage-specific expansions.

• Klíčová slova
• Giardia intestinalis, LECA, Puf superfamily proteins, RNA processing, RNA-binding protein,
• MeSH
• Eukaryota genetika   metabolismus   MeSH
• fylogeneze MeSH
• messenger RNA metabolismus   MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• RNA ribozomální 18S metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• proteiny vázající RNA MeSH
• RNA ribozomální 18S MeSH